All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Higher plants primarily use three methods to limit or decrease the growth of pathogens: 1) the hypersensitive or gene-for-gene response; 2) inducible responses such as systemic acquired resistance; and 3) via antimicrobial peptides.
Anti-microbial peptides act as a type of innate immunity which limits pathogen growth and spread. This mechanism may play an important role in a plant's natural defenses against pathogens. Thionins, defensins, and non-specific lipid transfer proteins (nsLTPs) are the most common plant proteins reported to have anti-microbial properties (reviewed in Garcia-Olmedo et al., 1995; Broekaert et al., 1997).
Most experiments utilizing plants transgenic for anti-microbial peptides have assessed the effects of the over-expression of endogenous or heterologous anti-microbial proteins. For example, transgenic tobacco plants with constitutive expression of the barley LTP2 protein (Molina and Garcia-Olmedo, 1997) or the barley alpha-thionin (Carmona et al., 1993) showed much reduced bacterial pathogen infection on leaf tissue. Over-expression of endogenous thionin genes in Arabidopsis thaliana (Epple et al., 1997) also resulted in reduced wilt symptoms upon infection by Fusarium oxysporum. Molina et al. (1997, Plant J. 12(3):669-675) report the expression of the barley lipid transfer protein LTP2 in transgenic tobacco. Some of these genes have been demonstrated to be inducible by pathogen infection (Molina et al., 1996).
Synergistic enhancement of anti-microbial properties has been demonstrated in vitro for a number of different anti-microbial proteins (Dubreil et al., 1998; Terras et al., 1993). The effects of multiple anti-microbial proteins may be expected to be at least additive.
There is a continuing need for alternative and supplementary methods of protecting plants from plant pests, including plant pathogens.
This invention provides for the control of pests via introduction of the puroindoline a gene (pinA) and the puroindoline b gene (pinB) into plant cells, plant tissues and plants. The constitutive protection offered by the PINA and PINB proteins, either singly or together, is surprising since these proteins are not part of an inducible defense found naturally in plants. Assessment of the inhibitory effect of the PINA and PINB proteins on pathogen growth on leaf tissue as demonstrated in the present invention is also unexpected since pinA and pinB are not expressed at all in normal leaf tissue (i.e., no puroindoline gene homologues are expressed in leaf tissue.). Prior to the present invention, no demonstration of in vivo anti-microbial properties has been demonstrated for the puroindolines. Thus, the present invention provides a new and important set of tools and methods for the protection of plants to pests which affect plant growth and yield.